lecture 22 Flashcards
(21 cards)
what do lncRNA lack?
how long are they?
abundance?
not a lot of open reading frames
> 200 nt, but no protein encoded
very common but low abundance!
LOW seq conservation
what is the chromatin status on random X inactivation?
active is euchromatic
inactive is entirely heterochromatic in each nucleus–> forms Barr body on the nucleus
lncRNA gene, Xist, is active and inactive where?
where is it located?
active on the heterchromic X chromosome, which is inactive
inactive on euchormic chromosome, which is active
XIC center of the chromosome–> it never leaves the nucleus and is never translated
how are scaffolds used in IncRNA to recruit proteins by Xist?
PRC2 role?
1) Xist is transcribed on inactive X chromosome
2) Xist coats the inactive X
3) Xist recruits PRC2 to inactive X
4) establishes H3K27me3 to repress genes
what are the fragments cut by dicer attached to?
what are the single strands produced by RISC called and what do they do?
bound to RISC—>which denatures dsRNA into single strands
guide strand stays bound to RISC
guide strands can
1) use compl base pairing to attach to mRNA–> degraded
2) RISC binds to compl mRNA–> blocks translation
3) directs chromatin modfying enzymes to nucleus
what is the structure of Dicer?
–dsRNA binding site– PAZ
–two RNase domains separated by the PAZ site
what is the purpose and examples of mRNA, housekeeping RNA and non coding RNA?
-mRNA: encoded by pol2 and involved in metabolism and protein encoding
-housekeeping RNA—involved in expression of all genes– tRNA, rRNA, snRNA
noncoding– perform various specific functions
what are functions of non coding RNA?
defense against virus and transposable elements
gene regulation– x chromosome inactivation, imprinting
telomere maitencne
why are RNA molecules able to perform so many functions?
contain genetic info in their nt sequence
can adopt complex structures
can be mobile
can interact w/ other nucleic acids and proteins
how did RNAi in petunias work?
chimer chalcone synthase showed reversible co suppression of genes in trans ways
what are RNAi?
how is it made?
what is the source of dsRNA?
a type of siRNA for defense
1) need initiator— virus, trans gene, transposable element
-exogenous or endogenous aberrant RNA
2) dicer will take the dsRNA and the endonuclease will cut RNA at the ends to make SiRNA
3) hairpin, comp RNA, RdRP
how is the RISC complex formed and what does it do?
1) ds siRNA is incorporated into argonaute protein (AGO)-> which denatures the siRNA so it is ss
2) siRNA and AGO form the RISC complex
3) RISC uses the ss SiRNA as a guide strand to sequence to complementary pair so that it can specifically silence certain stuff
what are the 2 outcomes of the RISC complex?
post transcriptionally: mRNA degregation via complementary binding to guide siRNA
on transcription: can go back to nucleus and find complementary DNA, which then will be methylated and forms HETEROCHROMATIN
how does tomato bushy stunt virus stop degradation by siRNAs?
1) dsRNA siRNA made against the ssRNA TBSV cut by dicer
2) P19 binds the siRNA, which binds and sequesters the siRNA, so RISC can’t bind and degrade the ssRNA from the TBSV virus
why and how are transposable elements silenced by siRNAs?
–are mobile, so they must be repressed to prevent proliferation
–are DS
-can form hairpins
-result from improper RNA processing and can use RdRT
1) loaded into dicer–> siRNA is generated + APO protein to make the RISC complex
2) RISC destroys TE mRNA and makes heterochromatin
how does CRISPR work? what is its analogous parts to RNAi?
1) have a PHAGE that introduces a spacer–> which has sequence we want
–spacer is like initiating RNA
2) transcription from the spacers , in which it is cut into mature crRNA
–similar to dicer
3) mature is like siRNA’s
4) Cas-RNA complexes form
–similar to putting the siRNA + the AGO to form the RISC
5) target destruction of complementary DNA
how is miRNA made?
1) initiator: transcript of miRNA which is transcribed by RNA pol 2
-dsRNA via hairpin
-only endogenous sources
-genes are encoded in the genome
2) drosha (RNase) cuts pre-miRNA into shorter fragments
3) exported to the cytoplasm
4) in cytoplasm, Dicer chops pre-miRNA into mature miRNA (-22 nt long)
Same for plants but only have dicer 1
how are genes regulated by miRNA?
1) guide strand loaded +AGO into RISC
2) looks for partially complementary regions in mRNA , usually 3’ UTR
3) based on complementary sequences:
-can prevents the ribosome form translating mRNA into protein (post transcriptional)
-RISC can cut mRNA –> degradation
how do lncRNA interfere with transcription via Airn?
Airn being transcribed binds to Pol2 and makes it fall off so it cannot bind mRNA
-or it can make pol 2 fall off even if transcription has already started
this is because even if mRNA transcription is started before lncRNA, just IncRNA being started will immediately stop mRNA from being transcribed
how does incRNa modify chromosome looping via ThymoD?
1)starts as both IncRNA and gene is methylated, so turned off
2) IncRNa is transcribed, which results in DNA methylation being lost in both the Thymol and the mRNA gene
3) CTCF will bind to the mRNA gene and loop will form
How is siRNA made?
-Dicer cuts dsRNA
-AGO protein in either RITS or RISC
-Passenger strand cleaved
Targets its target fully complementary
